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Regulation of the Stability of the Protein Kinase DYRK1A: Establishing Connections with the Wnt Signaling Pathway

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Regulation of the Stability of the Protein Kinase DYRK1A: Establishing Connections with the Wnt Signaling Pathway Regulation of the stability of the protein kinase DYRK1A: establishing connections with the Wnt signaling pathway Krisztina Arató TESI DOCTORAL UPF / 2010 Barcelona, November 2010 Regulation of the stability of the protein kinase DYRK1A: establishing connections with the Wnt signaling pathway Krisztina Arató Memòria presentada per optar al grau de Doctora per la Universitat Pompeu Fabra. Aquesta tesi ha estat realitzada sota la direcció de la Dra. Susana de la Luna al Centre de Regulació Genòmica (CRG, Barcelona), dins del Programa de Genes i Malaltia. Krisztina Arató Susana de la Luna A Pere, por haberme traído a Barcelona… Cover design by Luisa Lente (www.yoyo.es). Index Page Abstract/Resumen.................................................................................. 1 Introduction............................................................................................. 5 The protein kinase DYRK1A................................................................ 7 The DYRK family of protein ....................................................... 7 Structure and mechanism of activation of DYRK1A kinase........ 8 Regulation of DYRK1A expression ............................................ 10 Regulation of DYRK1A subcellular localization.......................... 11 Regulation of DYRK1A activity................................................... 13 DYRK1A as a regulator of signaling pathways........................... 14 The Notch signaling pathway........................................... 16 Receptor tyrosine kinase signaling.................................. 17 Hedgehog signaling......................................................... 19 Calcineurin-NFAT signaling............................................. 20 DYRK1A and disease................................................................. 22 Nemo-like kinase (NLK)........................................................................ 23 Regulation of NLK expression..................................................... 23 The subcellular localization of NLK............................................. 24 Mechanism of activation of NLK.................................................. 24 Physiological functions of NLK.................................................... 25 NLK as a regulator of signaling pathways................................... 26 NLK and disease......................................................................... 30 Wnt signaling pathway......................................................................... 31 Wnt ligands, biogenesis and extracellular transport................... 32 Receptors dictate Wnt activity..................................................... 33 Wnt--catenin signaling............................................................... 34 Objectives................................................................................................ 39 Materials and Methods............................................................................ 43 1. Plasmids............................................................................................ 45 1.1. Backbone vectors................................................................. 45 1.2. DYRK1A expression vectors................................................ 45 1.3. Other DYRKs expression vectors........................................ 46 1.4. NLK expression vectors....................................................... 46 1.5. -catenin expression vectors............................................... 46 1.6. Other plasmids..................................................................... 47 2. Techniques for DNA manipulation................................................... 48 2.1. DNA purification and sequencing......................................... 48 2.2. Cloning of a DNA fragment into an expression vector......... 48 2.3. Site-directed mutagenesis.................................................... 50 3. Cell culture........................................................................................ 51 3.1. Cell lines............................................................................... 51 3.2. Cell transfection.................................................................... 51 3.3. Cell treatments..................................................................... 51 3.4. Pulse-chase experiments..................................................... 52 3.5. Preparation of lentivirus stocks and infection....................... 52 4. Techniques for protein analysis........................................................ 53 4.1. Preparation of cell lysates.................................................... 53 4.2. Western blot analysis........................................................... 53 4.3. Immunoprecipitation assays................................................. 54 4.4. Immunofluorescence............................................................ 55 4.5. Purification of GST-fusion proteins....................................... 55 4.6. Pull-down assays................................................................. 56 4.7. In vitro kinase assays........................................................... 57 4.8. Mass spectrometry analysis................................................. 57 4.9. Reporter assays................................................................... 58 4.10. Protein quantification.......................................................... 58 5. Bioinformatic tools and statistical analysis....................................... 58 5.1. Search in the databases....................................................... 58 5.2. DNA sequence analysis....................................................... 59 5.3. Protein analysis.................................................................... 59 5.4. Statistical analysis................................................................ 59 Results..................................................................................................... 63 DYRK1A interacts with NLK................................................................. 65 The interaction between DYRK1A and NLK is independent of their kinase activities.................................................................................... 66 NLK interacts with all DYRK family members....................................... 67 The N-terminal region of DYRK1A has an NLK-binding site................ 68 NLK interacts with DYRK1A through its C-terminal region.................. 71 NLK phosphorylates DYRK1A............................................................. 73 NLK phosphorylation of DYRK1A affects neither enzymatic activity nor subcellular localization................................................................... 73 NLK co-expression induces a decrease in the accumulation of DYRK1A............................................................................................... 76 NLK induces DYRK1A proteasome-dependent degradation................ 77 NLK affects DYRK1A half-life through phosphorylation of multiple sites...................................................................................................... 79 The Wnt1/Wnt3a-Fzd1-HIPK2-NLK signaling cascade affects DYRK1A half-life................................................................................... 82 DYRK1A is a positive regulator of the transcriptional activity of a - catenin-dependent reporter.................................................................. 83 DYRK1A interacts with -catenin......................................................... 85 DYRK1A forms a ternary complex with the -catenin/TCF transcriptional complex......................................................................... 86 DYRK1A phosphorylates -catenin at multiple sites............................ 87 DYRK1A enhances -catenin-dependent transcriptional activity through phosphorylation of serine residues 552 and 675..................... 90 NLK inhibits the DYRK1A enhancement on the -catenin-dependent transcriptional activity........................................................................... 91 Discussion............................................................................................... 93 The protein kinases DYRK1A and NLK interact................................... 95 DYRK1A is a substrate of NLK............................................................. 98 DYRK1A plays a positive role on -catenin-dependent transcriptional activity................................................................................................... 101 Final remarks and future prospects...................................................... 105 Conclusions............................................................................................. 109 References............................................................................................... 113 Abbreviations.......................................................................................... 131 ABSTRACT Abstract DYRK1A is the most studied member of the DYRK family of protein kinases, because is one of the human chromosoma 21 proteins for which changes in gene dosage result in neuropathological alterations. DYRKs are activated by autophosphorylation on a tyrosine residue in the activation loop, a one-off event that takes place during translation. Accordingly, DYRK1A would be constitutively active once is synthesized. However, DYRK1A is extremely sensitive to gene dosage, and thus it is predictable that not only
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